前两期我们介绍了Sage系列产品在Small RNA、ChiP-seq、cffDNA和ctDNA测序中的应用，本期我们将介绍Sage系列产品在RAD seq和甲基化测序中的应用。

RAD-seq 相关应用

RAD-seq技术，是广泛用于遗传多样性测序技术的一种。利用限制性内切酶消化以降低基因组的复杂度，对特定酶切片段进行NGS高通量测序，实现基因分型的技术。

RAD-seq不仅实验操作简单，性价比高，更为重要的是他并不依赖参考基因组的信息，一次测序即可获得数以万计的多态性遗传标记，已广泛应用于生态学、遗传学、基因组学等研究领域。

在ddRAD-seq实验流程中，Pippin 系列全自动DNA片段回收仪，可以降低片段选择过程对相同位点在文库间代表性的影响，另外通过实验初期的监测，可以评估样品的酶切片段均匀度，排除转座子等高频出现的片段。

相关文献：

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4	Kim, Han-Na, et al. "The Origin and Invasion Pathway of Brown Rats Rattus norvegicus on Dok-Do Island Revealed by Genome-Wide Markers from 3-RADseq Approach." Animals 13.7 (2023): 1243.
5	Gargiulo, Roberta, Tiiu Kull, and Michael F. Fay. "Effective double‐digest RAD sequencing and genotyping despite large genome size." Molecular Ecology Resources 21.4 (2021): 1037-1055.
6	Goodall, Jake, et al. "RAD sequencing of common whelk, Buccinum undatum, reveals fine‐scale population structuring in Europe and cryptic speciation within the North Atlantic." Ecology and Evolution 11.6 (2021): 2616-2629.

甲基化测序（MeD-seq）应用

甲基化测序是一类专门分析DNA分子中胞嘧啶（C）甲基化水平的测序分析技术。其主要原理是通过将DNA中非甲基化的胞嘧啶转化为尿嘧啶（U），继而进行测序的技术手段。由于正常情况下胞嘧啶的配对碱基是鸟嘌呤（G），而尿嘧啶的配对碱基是腺嘌呤（A）。这样就可以在生成配对序列时产生不同的序列，进一步通过测序分析和参考链比较，以了解哪些部位发生了甲基化。

目前，减少代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing ，RRBS）覆盖了大部分的基因启动子和CpG岛，但是对CpG周围和其他有生物学意义的基因间区域的覆盖却不足。因此有越来越多的研究人员开发了增强减少代表性亚硫酸氢盐测序技术（Enhanced Reduced Representation Bisulfite Sequencing ，ERRBS）。ERRBS技术可以在在产生的数据中有覆盖更多的CpG，并增加了所有待测基因组区域的覆盖率，因此改善了RRBS在甲基化测序中的缺陷。其中在Garrett-Bakelman等人设计的ERRBS方法，通过利用Pippin Prep回收135-410bp的DNA片段，可以高效回收25ng以上的DNA并用于构建DNA文库，经过验证是一种可行的甲基化测序技术。

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